The invention relates generally to the isolation of natural L-xcex2-3-indolylalanine (L-xcex2-3) and the provision of natural amino acid mixtures enriched with natural L-xcex2-3. The L-xcex2-3 and amino acid mixtures containing L-xcex2-3 provide dietary therapeutic supplements for increasing the production of serotonin within the brain, thereby decreasing or eliminating undesirable physiological conditions brought about by a decreased brain serotonin level.
In order for multicellular organisms to function, it is necessary for the cells of a body to communicate with each other. In this way, it is possible to coordinate responses as required to constantly adjust to a continually changing external and internal environment [1]. This communication process is dependent on two operating systems, i.e., the nervous system in which signals or messages are transmitted, and hormones which are secreted and transported to adjacent or distant tissues. Both of these systems initiate specific physiological actions dependent on the particular type of cell that is activated.
The first step in the transmission of a brain signal is the synthesis of a chemical molecule called a neurotransmitter. Of the many brain neurotransmitters that have been identified, several are not synthesized de novo in nerve terminals, but rather are the result of a series of enzymatic reactions which modify a precursor molecule, usually an amino acid. After the molecules of the neurotransmitter have been biosynthesized, they are stored in the axon terminals of pre-synaptic nerve fibers in tiny membrane-bound sacs called synaptic vesicles which serve to protect the neurotransmitter molecules until they are used.
Serotonin is a neurotransmitter which the brain utilizes to send messages (electrical impulses) from one brain cell to another. Brain levels of serotonin have been shown to be involved in diverse physiologic processes, the most studied being sleep, appetite, mood, and pain threshold. Biochemical disturbances in the brain resulting in reduced levels of serotonin have been linked to insomnia [2][3], excessive appetite and weight gain [4][5], clinical depression, aggressiveness [6][7][8], and lowered pain threshold [9][10][11]. The latter abnormality results in chronic, intractable pain that generally is refractory to treatment by conventional medications.
The neurotransmitter serotonin is synthesized in the brain from the amino acid L-xcex2-3. L-xcex2-3 cannot be made in the body. L-xcex2-3 must be introduced into the body from an outside source, such as from protein in food or as a dietary supplement. Along with the other amino acids present in the blood stream (which are absorbed from the small intestine from hydrolytic digestive processes in the gastrointestinal tract), L-xcex2-3 is carried to the brain. In the brain, a very selective process then takes place prior to the formation of serotonin.
In order for L-xcex2-3 to be converted to serotonin, L-xcex2-3 must first cross a separating mechanism that exists between the blood vessels in the brain and the brain proper. For L-xcex2-3 to pass from the circulating blood through the blood/brain barrier, a transport mechanism in the form of a carrier protein is required. The primary function of this mechanism is to isolate L-xcex2-3 from the majority of other amino acids circulating in the blood and, then, literally to transport L-xcex2-3 across this selective blood/brain barrier into the brain. There, a two-step enzymatic process converts the L-xcex2-3 first to 5-hydroxy-L-xcex2-3 and then to serotonin.
L-xcex2-3, however, is not the only amino acid carried by this transport mechanism. Five other amino acids, termed large neutral amino acids (LNAAs), are carried as well. LNAAs include phenylalanine, tyrosine, leucine, isoleucine, and valine. L-xcex2-3 not only has to compete with these LNAAs for access to the transport mechanisms, but L-xcex2-3 also has a lower affinity for the carrier system than do the LNAAs. Of the five LNAAs, phenylalanine is the most tightly bound to the transport protein and is therefore the most detrimental to the transport of L-xcex2-3 across the blood/brain barrier. To complicate this situation further, L-xcex2-3 in foods is present in lower amounts than the LNAAs, particularly in animal proteins. All of these factors converge to limit the amount of L-xcex2-3 that gets through to the brain to be finally converted into serotonin.
It is known that dietary supplementation with L-xcex2-3 increases the blood level of L-xcex2-3 and facilitates the passage of L-xcex2-3 across the blood/brain barrier into the brain. The increased amount of L-xcex2-3 in the brain permits a greater amount of L-xcex2-3 to be converted to serotonin. There are, however, numerous conditions that can interfere with and decrease the amount of L-xcex2-3 that normally passes through the blood/brain barrier into the brain each day. The primary factor that controls the degree to which L-xcex2-3 is transported across the blood/brain barrier is the ratio of L-xcex2-3 to LNAAs that is present in the blood going to the brain. At a lower-than-normal L-xcex2-3 to LNAA ratio, the number of molecules of L-xcex2-3 present at the blood/brain barrier is less than normal. The LNAAs, which are normally present in larger numbers than L-xcex2-3, then overwhelm the L-xcex2-3 by monopolizing the majority of the transport carriers, and even less L-xcex2-3 passes across the blood/brain barrier and into the brain as compared to the number of LNAAs that are passed across the barrier. In attempting to correct this improper L-xcex2-3/LNAA ratio, it was found that increasing dietary protein intake in order to add more L-xcex2-3 to the system can result, paradoxically, in an even greater derangement of the L-xcex2-3/LNAA ratio because of the simultaneous greater intake of LNAAs over the intake of L-xcex2-3.
One means by which the L-xcex2-3/LNAA ratio abnormality can be treated is by the administration of L-xcex2-3 without the accompanying presence of the LNAAs, especially without the presence of phenylalanine. This administration of L-xcex2-3 serves to increase the L-xcex2-3 portion of the circulating L-xcex2-3/LNAA ratio, increase the amount of L-xcex2-3 which will be transported across the blood/brain barrier into the brain, increase the L-xcex2-3 pool in the brain, and increase the rate of conversion of L-xcex2-3 to serotonin.
Prior to 1989, L-xcex2-3 was available to consumers as a dietary supplement and could be purchased freely. Studies on the oral administration of L-xcex2-3 under proper dietary conditions that provided a supplementary intake of this particular amino acid showed that supplemental L-xcex2-3 helped to correct an improper L-xcex2-3/LNAA ratio in the brain. This increased level of brain L-xcex2-3 directly produced an increased brain serotonin level which was associated with a reduction or elimination of serotonin-deficiency syndromes.
In the late 1980""s, none of the L-xcex2-3 available in nationally-marketed preparations was produced in the United States. All of the L-xcex2-3 used in the United States was imported from Japan. In 1989, the importation and sale of L-xcex2-3 in the U.S. was halted by the U.S. Food and Drug Administration (FDA) as a result of a highly toxic contaminant that was found in batches of L-xcex2-3 made by a bacterial fermentation process used by one particular Japanese company. To date, the importation of L-xcex2-3 into the U.S. and the sale of all imported L-xcex2-3-containing products has not resumed. L-xcex2-3 is still unavailable to the public because of the difficulty in excluding possible toxic compounds which could be generated in L-xcex2-3 produced by fermentative processes.
Natural L-xcex2-3 is the only substance which will increase brain serotonin in a normal physiological manner. A need exists for natural L-xcex2-3 which can serve as a direct precursor for the synthesis of serotonin in the brain, serve as a source of serotonin which is free of the many side effects encountered with the use of serotonin-enhancing medications, and is obtained in a manner which ensures that no potentially toxic compounds are produced by the microorganism which produces the L-xcex2-3 as a part of its metabolic cycle.
The invention relates to L-xcex2-3 as a naturally-occurring amino acid found in all common dietary proteins; as the amino acid naturally used by the body to produce serotonin in the brain; and as obtained simply and directly from natural dietary proteins, thereby being free of biologically produced contaminants.
Thus, primary objects of the invention include separation of the amino acid L-xcex2-3-indolylalanine from a natural source of a mixture of amino acids, preferably enzymatic or other natural protein hydrolysates containing mixtures of free amino acids; preparation of an amino acid fraction from the aforementioned L-xcex2-3 and an amino acid mixture (obtained during the aforementioned separation) that is free of aromatic amino acids, particularly phenylalanine; and preparation of highly enriched mixtures of L-xcex2-3 and one or more non-aromatic amino acids, i.e., mixtures having a concentration of L-xcex2-3 in an amount greater than that which occurs naturally.
More particularly, the invention is directed to providing processes, and compositions based on compounds obtained by such processes, for (1) the separation, as a group, of aromatic amino acids, including L-xcex2-3, from an amino acid mixture, the mixture preferably being obtained by the hydrolysis of common proteins normally used for dietary purposes; (2) the removal of L-xcex2-3 from the mixture of aromatic amino acids obtained in (1); and (3) producing mixtures of one or more non-aromatic amino acids with the recovered L-xcex2-3 in various proportions. All components isolated or recovered in the process of the invention are provided in a form suitable for further use.
The invention provides a process whereby a mixture of natural amino acids containing substantially all of the natural amino acids (i.e., glycine, the L-forms of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, hydroxyproline, L-xcex2-3, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine) in the form of free amino acids is separated into three fractions containing, respectively, (a) substantially all of the non-aromatic amino acids originally present, i.e., alanine, arginine, asparagine, aspartic acid, cysteine (in equilibrium with the dimeric cystine), glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and valine; (b) the monocyclic aromatic amino acids phenylalanine and tyrosine; and (c) L-xcex2-3, the only amino acid of the group that possesses as a part of its structure two fused aromatic rings.
The invention further provides a range of mixtures of fraction (a) with fraction (c) while specifically eliminating fraction (b) which is deleterious to the desirable physiological actions of L-xcex2-3. These mixtures including one or more aliphatic amino acids and L-xcex2-3 are possessed of highly desirable physiological properties useful in the therapeutic relief of human suffering related to and caused, at least in part, by a relative deficiency of L-xcex2-3.
The present invention involves the discovery that when a mixture containing phenylalanine, tyrosine, and L-xcex2-3 in the presence of mixed aliphatic amino acids is dissolved in water and applied or exposed by contact to a porous, wettable polymeric resin having attraction for aromatic rings of amino acids but little or no attraction to aliphatic amino acids at the natural pH of the solution, that the mixed aromatic amino acids adsorb selectively to the resin and can thereafter themselves be selectively and sequentially desorbed. This is accomplished by first taking a natural amino acid-containing mixture or source, preferably an enzymatic or other natural protein hydrolysate, dissolving it in water, and passing it over a polymeric resin having properties as described above, preferably a non-ionic cross-linked polystyrene, in order that the three aromatic amino acids will be selectively attracted to the resin and the aliphatic amino acids carried away in the fluid carrier. The resin is thereafter washed with deionized water to remove any residual non-aromatic or aliphatic amino acids which, while having essentially no affinity for the resin, may be physically associated with but not bound by attractive forces to the resin. Then the resin is subjected to serial elutions with a suitable dilute acid and dilute base. Suitable acids are short chain aliphatic acids having molecular weights no greater than 88.10 daltons and a Ka between from 1.77xc3x9710xe2x88x924 to 1.34xc3x9710xe2x88x925 (pKa of between from 3.75 to 4.87) at 25xc2x0 C. Suitable bases are ammonia and short chain aliphatic primary secondary or tertiary amines having molecular weights no greater than 101.19 daltons and a Kb between from 1.26xc3x9710xe2x88x923 to 1.8xc3x9710xe2x88x925 (pKb of between from 2.90 to 4.74) at 25xc2x0 C. A preferred acid and base is dilute acetic acid and dilute ammonium hydroxide, respectively, from the standpoint of both function and economy. The resin is first washed with the dilute acid to selectively desorb phenylalanine and tyrosine from the resin while leaving L-xcex2-3 adsorbed to the resin. The resin is then washed with deionized water to remove the acid. Thereafter, the resin is washed with the dilute base to displace the L-xcex2-3 from the resin. Both the acid and the base will form weak salts with amino acids, which readily decompose upon gentle heating. Since both the acid and the base are very volatile and amino acids are not, the solutions can readily be heated under vacuum to remove water and either the acid (from the phenylalanine plus tyrosine fraction) or the base (from the L-xcex2-3 fraction) leaving behind the free amino acids in substantially pure form. This evaporative concentration when performed on the L-xcex2-3-containing solution provides a dry, non-hygoscopic powder while removing excess base. Alternatively, the L-xcex2-3 is recovered by crystallization. The serial elutions with dilute acid and dilute base allow the L-xcex2-3 first to be held to the polymeric resin while removing phenylalanine and tyrosine and, thereafter, isolating the L-xcex2-3 from a major amount of other amino acids, as well as residual dipeptides, in the starting hydrolysate mixture. It is particularly advantageous to remove phenylalanine from L-xcex2-3 since, as described above, phenylalanine is strongly competitive with L-xcex2-3 in the key systems which transport L-xcex2-3 to the brain.
All of the examples herein are based upon the use of an enzymatic hydrolysate of casein. An enzymatic hydrolysate of soy protein is also useful and is highly preferred since it contains twice as much free L-xcex2-3 as does casein hydrolysate. Other natural protein hydrolysates are necessarily also useful. The particular hydrolysate used will depend on availability. The protein hydrolysate used can be xe2x80x9cconcentratedxe2x80x9d in the sense that a higher amount of protein hydrolysate is present to the amount of water when put in solution as compared to conventional preparations. The protein hydrolysate is generally present in an amount of 1-30% by wt. of the aqueous solution containing the protein hydrolysate. A preferred range for the present invention is 5 to 16% by weight. Most food-acceptable protein sources contain L-xcex2-3 at about 0.5 to 1.5% by weight of the contained protein. The invention serves to concentrate the L-xcex2-3 from these sources to a range of about at least 10 to 75%.
Suitable polymeric resins for use in the invention are porous wettable polymeric resins which have little attraction to aliphatic amino acids but are attractive to aromatic rings of amino acids. The attraction to the aromatic rings of amino acids is believed to be based on the polymeric resin having attractive van der Waals interaction due to the .pi.-electrons of the polymer with the .pi.-electrons of the aromatic rings of the amino acids. A preferred polymeric resin suitable for use in the present invention is a nonionic cross-linked polystyrene such as sold under the name AMBERLITE(copyright) XAD-4 resin sold by Rohm and Haas Company. Other polymeric resins also sold by Rohm and Haas which are suitable for use include, but are not limited to, the following: Amberlite XAD-16, Amberlite XAD 1180, Amberlite XAD-2000, Amberlite XAD-2010, Diaion HP20, Diaion HP20SS, Sepabeads SP20MS, Amberchrom CG-71, Amberchrom CG-161, Amberchrom CG-300, Amberchrom CG-1000, Ambersorb 563, Ambersorb 575, Ambersorb 348F, and Ambersorb 572. The resins are preferably present in particulate form, most preferably in the form of porous beads.
Suitable acids, in dilute form, suitable for use include, but are not limited to, acetic acid, formic acid, propionic acid, and butyric or i-butyric acid. As stated above, dilute acetic acid is preferred.
Suitable bases, in dilute form, suitable for use include, but are not limited to, ammonia (in the form of ammonium hydroxide), trimethylamine, triethylamine, (CH3)2NH, (C2H5)2NH, CH3NH2 and CH3CH2 NH2. Ammonium hydroxide is preferred.